Abstract
Specific carbohydrate structures in glycoconjugates play important roles in biological systems. Glycosyltransferases catalyze the synthesis of different disaccharide linkages in oligosaccharides and a different glycosyltransferase is required to synthesize each type of glycosidic linkage. Little structural and functional information is currently available regarding glycosyltransferases. In this study, we investigated the catalytic mechanism and structure-function relationships in two galactosyltransferases: beta1,4 galactosyltransferase and beta1,3 galactosyltransferase, which catalyze the synthesis of a Galp1,4GlcNAcbeta-OR structure and the alpha-Gal epitope (Galalpha1,3Galbeta-GlcNAcbeta-OR), respectively.A truncated soluble form of the recombinant bovine beta1,4 galactosyltransferase (rbeta4GT-1) was expressed on a large scale with E. coli. Mutations were induced to a highly conserved region of sequence that includes a cluster of acidic residues. Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the structure of beta4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor.A soluble form of bovine alpha1,3 galactosyltransferase (ralpha3GT) that was expressed in E. coli. OGT utilizes a sequential mechanism and also catalyzes T-TDP-galactose hydrolysis at a low rate, showing that the LJDP to galactose bond is cleaved in the absence of acceptor substrate. Mn2+ has been considered as the OGT cofactor in previous studies. Our steady state kinetic studies showed the presence of low and high affinity binding sites for Mn2+ and a high affinity binding site for Zn2+, which may be the biological relevant OGT cofactor. The role of the metal cofactor was investigated through the effects of perturbing metal activation by substitution Mn2+ with CO2+ and Zn2+.A new homologue (designated as LZH) of lysozyme and alpha-lactalbumin (a regulatory protein for beta4GT-1) was expressed, purified and characterized. A homology-based model and other sequence-derived properties indicate that LM shares some key structural features with alpha-lactalbumin. (Abstract shortened by UMI.)